For successful gene transfer to primitive hematopoietic cells several requirements need to be achieved. These include identification of the desired target cell population, identification of the appropriate vector to be used, and achieving desired levels of gene expression. To date, successful gene transfer in human subjects remain problematic. To address these problems as well as important safety issues, studies in non-human primates are being undertaken to optimize gene transfer to nonhuman primate hematopoietic cells prior to human clinical studies. Vectors that have been evaluated over the past year were constructed to express the reporter genes encoding the enhanced green flourescent protein (EGFP) and the neomycin resistance gene. Transduction conditions employed the RGD-containing fibronectin fragment, RetroNectin (CH-296) and a variety of recombinant hematopoietic growth factors which included stem cell factor, interleukin-6, megakaryocyte growth and differentiation factor (MGDF or thrombopoietin) and the human Flt-3 (fms-like tyrosine kinase) ligand in either serum containing and serum free media. Retroviral vectors include Moloney murine leukemia-based vectors and a third generation human immunodeficiency virus type-1-based lentiviral vector. Third generation lentiviral vectors were pseudotyped with the vesicular stomatitis virus G-protein. Our efforts over the past year have primarily involved comparisons of cytokine therapies, G-CSF alone and the combination of G-CSF and SCF,in order to optimize mobilization of target cell populations that can be effectively transduced with a retroviral vector and contribute to long term hematopoietic recovery of a myeloablated animal. In addition, genetic tracking continues to be performed on cells that have been effectively transduced with a retroviral vector in order to determine their contributions to hematopoietic lineage recovery. Persistent multiple lineage marking using clinically feasible protocols has been achieved ranging from 5-15%. Efforts are being made to extend this level of marking to stem cells derived from other tissues besides BM and cytokine mobilized PB, such as muscle and fat. In addition, several studies have been initiated to study the immune status of the non-human primates following transplant. Prolonged expression of reporter genes, such as EGFP or the bacterial neomycin resistance gene, appears to be associated with tolerance induction associated with transplant radiation. Phenotypic and functional analysis of cells following hematopoietic reconstitution are being evaluated. Despite these successes, questions remain. How can consistent high levels of expression be obtained using therapeutic genes? Can other stem cells either derived from bone marrow or other easily accessible tissues be targeted to assist in either the contribution or repair of other organs? These questions are being addressed currently through the evaluation of vectors using novel reporter genes such as the Herpes viral thymidine kinase gene, further delineation of the nature and clonality of the population contributing to the reconstitution using genetic tracking methodologies, and with the initiation of new studies to isolate, characterize, and identify cell populations that may contribute to organogenesis or the repair of damaged tissues such as the heart.